With the implementation and progress of the Human Genome Project, life science research has entered the post-genome era. In this era, life science turns to focus on proteins because the whole-genome sequence information is insufficient to explain or speculate various life phenomena, but proteins play a significant role in executing the physiological functions and directly reflect life phenomena, hence researches on the structure and function of proteins will directly explain the mechanism of changes of life under physiological or pathological conditions before and after drug intervention.
In the proteomic analysis of a biological sample, in order to determine the types of proteins contained in the biological sample or find out a target protein of interest, first of all, it is necessary to separate proteins in the biological sample. The existing two-dimensional gel electrophoresis analysis method can separate proteins in a biological sample in two dimensions. Typically, the method involves firstly separating proteins in the first dimension by isoelectric focusing electrophoresis (first-dimensional electrophoresis) based on the difference in protein isoelectric point, and then separating the proteins resulting from the first-dimension separation in the second dimension by polyacrylamide gel electrophoresis (second-dimensional electrophoresis) based on the difference in protein molecular weight. After the separation of second-dimensional electrophoresis, the separated proteins on the gel are stained, so that the proteins in the gel are visualized in the form of protein spots. Due to the differences in isoelectric point and molecular weight, different proteins are located in different positions of the gel. For a biological sample, there are tens of thousands of protein spots or even more. In order to identify proteins in the protein spots, it is necessary to excise the gel containing the protein spots (i.e., gel excision). Then the proteins are digested in the gel (i.e., in-gel digestion) to form peptide mixtures, and the peptide mixtures are extracted. Finally the peptide mixtures are made into sample targets (i.e., target preparation) by different mass spectrometry (MS) ionization methods, followed by MS analysis to obtain MS information of the proteins, such as peptide mass fingerprinting and peptide sequence tag. Thus, before MS analysis, the proteins separated by the second-dimensional electrophoresis need to undergo the sample preparation mentioned above, including staining, gel excision, in-gel digestion, peptide mixture extraction and target preparation. All the protein spots to be detected need to undergo the steps of gel excision, in-gel digestion, peptide mixture extraction and target preparation, such operation is feasible for the detection of a small amount of protein spots. However, as a biological sample contains a huge number of protein spots, it is time-consuming and laborious for the sample preparation of each protein spot. Even with automatic gel excision instruments, automatic enzymatic digestion instruments and automatic target making instruments, it is difficult to treat all protein spots on the gel one by one. Thus, it is urgent for proteomic analysis of biological samples to simplify the operations of second-dimensional electrophoresis and pretreatment before MS analysis and after electrophoresis.
Biochip technology provides the possibility, for example, microfluidic chip technology has been widely used in the field of proteomic analysis: a combination of isoelectric focusing and capillary electrophoresis (A. E. Herr et al., Anal. Chem., 75, 1180-1187, 2003), a combination of isoelectric focusing and capillary electrophoresis (Y. Li et al., Anal. Chem., 76, 742-748, 2004), and an integration of capillary electrophoresis, fractionation, solid phase extraction and electrospray ionization (ESI) (Q. Y. Lu, J.-B. Bao, D. J. Harrison, 11th Int. Conf. Miniatur. Syst. Chem. Life Sci., p. 44-46, 2007), etc.
However, at present, these attempts still do not meet the need for rapid preparation of a large number of samples in proteomic analysis, and a more practical microchip technology is required for proteomic analysis.